JP2012531924A - Introduction of DNA into plant cells - Google Patents

Abstract

The present invention provides means and methods for conveniently and efficiently introducing foreign genetic material into plant cells. In particular, the present invention combines seed priming and virus-based DNA constructs for efficient introduction of heterologous DNA into plants.
[Selection] Figure 1

Description

  The present invention relates to the field of plant molecular biology and specifically to means and methods for conveniently and efficiently introducing exogenous genetic material into plant cells.

  Plants having one or more expressible heterologous genes have various potential advantages. Such plants with gene expression cassettes are, for example, herbicide-resistant, insecticide-resistant or insect-resistant; resistance to stress; increased flavor and / or shelf life of fresh products (fruit, vegetables, seeds, etc.); and Useful plant endogenous and / or external proteins that are consumed by humans and / or animals or used as raw materials in various industries (cosmetics, pharmaceuticals, dietary supplements, foods, paper, fibers, etc.), It can have one or more genes that give the desired trait, including being able to amplify the synthesis of sugars, fatty acids or secondary metabolites.

  Current transformation techniques provide the opportunity to design plants with the desired traits, and significant advances in plant transformation have occurred in recent years. The most common technique is mediated by Agrobacterium tumefaciens. Other methods of transformation are direct DNA transfer including microinjection, electroporation, particle bombardment and viral vectors (Birch RG, 1997, Annu. Rev. Plant Physiol. Plant Mol. Biol., 48: 297-326). With the exception of viral vectors, application of the above technique results in the invasion of foreign DNA into the treated cells / tissue, its integration into the genome of the treated plant, and regeneration of the transgenic plant. However, in many major cereal plants, there are still not easy genotype restrictions. Transformation of some agriculturally important crop plants continues both to be difficult and time consuming. In addition, in all of the above techniques, transformation is performed using vegetative tissue (including embryos), transformation efficiency is poor, markers for selection are required, and transformed cells or The rate of successful plant regeneration from tissues is quite low.

  The integration of foreign DNA into the plant genome is not always desirable, especially when genetically modified (GMO) plants provoke environmental and political disputes. In these cases, a method is needed that allows the expression of heterologous genes that are not integrated into the host genome.

  International (PCT) Patent Application Publication No. WO2007 / 141790 by some of the inventors of the present invention is from one plant cell to another plant cell within a treated plant without causing pathological viral symptoms. Disclosed are modified geminivirus-type constructs that can spread and simultaneously introduce foreign DNA into plant cells. Although foreign DNA is expressed in plant tissue, the foreign DNA is not integrated into its genome. The construct (also shown as an expression vector, IL-60 is an example) is a discontinuous encoding a heterologous polynucleotide sequence that interferes with the geminivirus replicase gene, geminivirus replicase or replicase-associated protein. Nucleic acid sequences so that they are arranged adjacent to each other.

  Seed priming is a process for treating plant seeds that causes seeds to germinate faster and more uniformly when seeded or when planted compared to untreated plant seeds. It is a process that enables In addition, priming provides protection and / or optional concurrent treatment with fungicides / insecticides / fertilizers or other chemicals that facilitate germination, rooting and seedling establishment (Eg, coating, pelletizing, coloring as a trademark, etc.).

  Priming allows the seed to absorb enough water, allowing its pre-emergence metabolic process to begin, and then stopping the metabolic process at that stage. The amount of water absorbed (with or without beneficial chemicals) must be carefully controlled. This is because too much will simply allow the seeds to germinate and too little will result in seed senescence. Once the correct amount of water has been absorbed, the seeds can be sown or dried to return to their original moisture content for storage. Primed seeds usually germinate and root more quickly and uniformly, especially under non-optimal conditions, and seedling seedling vitality is typically greater than non-primed seeds. Greater than. Benefits obtained by priming, such as germination rate and germination uniformity, are usually attributed to the initiation of DNA repair processes, protein hydrase activation, and further processes that occur in the early stages of germination. .

  The three main techniques used for controlled moisture uptake include priming with aqueous solutions, priming with hydrated soil granulation systems, or priming with controlled hydration with water. . Priming with a solution is an osmotic potential that allows seeds to be immersed in an osmotic solution, typically limited water swell, and is insufficient for complete hydration and seed germination Based on immersing in a PEG solution characterized by an osmotic potential. In the alternative, the same effect is achieved by mixing the seed with a hydrated absorbent medium such as clay or peat (see US Pat. No. 4,912,874). Controlled hydration with aqueous solutions can be achieved, for example, by utilizing a semi-permeable membrane to mediate water transfer from a given osmotic solution to the seed (US Pat. No. 5,992,091). Priming is performed under various temperature and aeration methods (eg, stirring, agitation, bubbling, etc.) using any of the techniques for controlled moisture uptake (Taylor AG et al., 1998, Seed Science). Technology, 8: 245-256).

  Combining the general transformation techniques described above herein with seed priming is disclosed. For example, US Pat. No. 6,646,181 is a method for introducing a gene into a plant, wherein the plant developmental stage is synchronized at a stage where a large amount of 4C DNA is contained in the plant seed, and the seed cell is transfected. However, synchronizing the developmental stage may include mixing a particulate soil matrix material with a seed priming amount of water, with aeration of the seeds, and a substantial number of seed cells in the cell cycle. Disclosed is a method comprising performing at a time and temperature sufficient to cause the desired stage to be reached.

  US Patent Application Publication No. 2006/0005273 discloses corn explants suitable for transformation. The explant contains corn seeds that are divided in half along the length, but the division exposes the scutellum, coleoptillar ring and shoot apical meristem (each of which is independent) And suitable for transformation). By priming the seed before division together with either a callus priming medium or a shoot priming medium, the callus induction frequency and the shoot induction frequency after transformation are increased.

  US Patent Application Publication No. 201300154083 discloses compositions and methods for screening, identifying, selecting, isolating and / or regenerating targeted integration events using seed priming. Seed priming results in the identification of seeds that have stably incorporated site-specific recombinase-mediated integration of the selectable marker into their genome at a target locus that is operably linked to a promoter active in the seed.

  These combination methods use known methods of transformation and aim to increase the efficiency of foreign gene integration into the plant genome. As described hereinabove, available methods have limitations with respect to applicability and efficiency. In addition, the integration of foreign DNA into the plant genome is not always desirable.

  It has been recognized that there is a need for means and methods for conveniently and efficiently introducing DNA into plant cells that further enable efficient plant regeneration, and such means and methods are It would be very convenient to have.

  The present invention provides means and methods for the convenient and efficient delivery of heterologous DNA to plant cells, parts, tissues, organs or whole organisms, especially to seed embryo cells inside intact seeds. provide. The seed with the introduced heterologous DNA grows easily into a mature plant under standard growth conditions without the need for regenerative culture and enrichment conditions.

  The present invention provides, in part, that the introduction of heterologous DNA into plant seeds during the period of seed priming by supplementing the priming medium with a virus-based DNA construct, specifically an expression construct based on geminivirus. Based on the unexpected discovery that it is possible. As exemplified herein below, the present invention now includes in a priming medium a geminivirus-type construct, referred to as IL-60, which is a discrete protein encoding a geminivirus replicase or replicase-associated protein. Complementing nucleic acid sequences (including heterologous polynucleotide sequences arranged adjacent to each other) shows that DNA construct incorporation, replication, and asymptomatic expansion in seed cells.

  The teachings of the present invention are for plant cell transformation or introduction of foreign DNA in that the foreign DNA is introduced into an intact seed embryo that is naturally capable of developing into a complete plant. This is an advantage over previously known methods. The teachings of the present invention for DNA introduction do not involve damage to cells (such as occurs when direct DNA transfer, particularly direct DNA transfer by impact) is used, or damage to the functional expression of cells. . Moreover, when geminivirus-based expression constructs are used, heterologous DNA is not incorporated into the plant genome, which again causes cell cycle arrest and apoptosis or programmed cell death, albeit again. Prevents wounds associated with the introduction of DNA into cells. The simple method of the present invention can be used with the seeds of any plant of interest, is rapid and is very efficient. When the DNA construct used is such that heterologous DNA is not incorporated into the plant genome, the plant expresses the desired product but is not defined as genetically modified, so the method of the present invention Even more convenient.

  Thus, according to one aspect, the present invention provides a method for introducing heterologous DNA into at least one cell of a plant seed embryo, wherein the plant seed is subjected to conditions that allow priming. Contacting with a priming medium containing a viral DNA construct comprising, thereby obtaining a seed embryo comprising heterologous DNA.

  According to some embodiments, the method further comprises providing suitable conditions for subsequent seed germination and growth to obtain a plant comprising heterologous DNA or a portion thereof.

  According to some embodiments, the viral DNA construct comprises a viral gene or portion thereof that allows replication of the construct and asymptomatic expansion into neighboring plant cells.

  According to an exemplary embodiment, the DNA construct is a geminivirus type construct. According to these embodiments, the construct comprises heterologous DNA flanked by discontinuous nucleic acid sequences encoding a geminivirus replicase or replicase-associated protein.

  According to other embodiments, the construct further comprises a polynucleotide sequence encoding a modified geminivirus coat protein (CP). According to an exemplary embodiment, the polynucleotide encoding the modified geminivirus coat protein comprises a mutation or deletion in the nucleotide encoding the 100 amino acids at the N-terminus.

  According to a further exemplary embodiment, the expression construct further comprises a polynucleotide sequence encoding a modified geminivirus V2 protein.

  According to a further exemplary embodiment, the expression construct further comprises a polynucleotide sequence encoding a modified geminivirus C4 protein. According to these embodiments, the modified geminivirus C4 protein comprises a mutation or deletion. According to some embodiments, the expression construct further comprises a bacterial polynucleotide sequence.

  According to some currently preferred embodiments, the geminivirus is tomato yellow leaf curl virus (TYLCV). According to these embodiments, the geminivirus type construct is selected from the group consisting of IL-60 having the nucleic acid sequence set forth in SEQ ID NO: 1 and IL-60-BS having the nucleic acid sequence set forth in SEQ ID NO: 2. Is done.

  According to some embodiments, the DNA construct is designed as an expression construct such that heterologous DNA is expressed in plant cells. According to these embodiments, the DNA construct further comprises at least one regulatory element selected from the group consisting of an enhancer, a promoter and a transcription termination sequence.

  According to a further embodiment, the DNA construct further comprises a marker for identifying seeds and / or plants containing the heterologous DNA.

  The heterologous DNA can encode any desired product, including peptides, polypeptides, proteins and RNA. The protein or RNA can be of plant origin, or of other origins, including but not limited to bacterial and mammalian origin. According to some embodiments, the DNA encodes an inhibitory RNA selected from the group consisting of antisense RNA, dsRNA, siRNA, etc., that results in silencing of expression of the target gene. According to other embodiments, the heterologous DNA may be desirable agronomical, including but not limited to resistance to biological or abiotic stress, increased yield, increased harvest characteristics and preferred growth patterns. A trait encodes a product conferred by its expression. According to a further embodiment, the encoded protein product is useful in the cosmetic or pharmaceutical industry. According to yet a further embodiment, the encoded protein enhances production of a desired metabolite in plant cells and plant tissues.

  According to yet another embodiment, the heterologous DNA does not encode the desired product, but is a marker for seed origin, for example, to prevent illegal distribution of proprietary seeds, plants and tissues. Or merely as a label for plants grown from said seeds.

  Heterologous DNA can be expressed transiently in cells and cells derived from the cells, or the heterologous DNA may be incorporated into the cell genome. Heterologous DNA can be present in the cytoplasm of a plant cell, in its organelle, or in the nucleolus as an integrated or non-incorporated DNA sequence.

  According to some currently preferred embodiments, the DNA construct does not incorporate heterologous DNA into the cell genome, but the construct replicates and expands in plant cells grown from seeds containing the heterologous DNA. Is a geminivirus-type construct designed to

  Any priming system and priming conditions as known to those skilled in the art can be used in accordance with the teachings of the present invention, including systems containing aqueous solutions with sufficient osmotic potential, and systems containing solid particles. it can. The water potential of the medium allows for water uptake, which is insufficient for complete seed water uptake and swelling, and only allows for the early stages of germination, not rooting of radicles (complete germination). Such systems and methods are typically selected according to the plant seed species.

  According to a further embodiment, the present invention provides a seed produced by the method of the present invention, comprising a viral DNA construct comprising heterologous DNA, and a plant or a part thereof produced from said seed.

  According to some embodiments, the plant seed is of monocotyledonous plant origin. According to another embodiment, the plant seed is of dicotyledonous origin.

  Other objects, features and advantages of the present invention will become apparent from the following description and drawings.

FIG. 1 demonstrates the effect on seed priming on germination uniformity.

FIG. 2A is an illustration of a geminivirus-type construct used in the seed priming experiments described herein. FIG. 2A: IL-60-BS. FIG. 2B is an illustration of a geminivirus type construct used in the seed priming experiments described herein. FIG. 2B: pIR-GUS.

FIG. 3 shows the presence of GUS sequences detected by PCR in the tomato true leaves after seed priming in the presence of geminivirus type constructs. A dotted arrow and a black arrow indicate the position of the 1500 bp size marker and the position of GUS (about 1800 bp), respectively (lane 1). Lanes 8 and 12 are negative controls, respectively, PCR obtained from plants grown from untreated seeds and PCR obtained from plants derived from water-swollen seeds without a DNA construct. .

Figures 4A-4B show cross sections of tomato leaves stained for GUS activity after seed priming in the presence of IL-60-BS and pIR-GUS (Figure 4B, 40x magnification).

Definitions The term “priming” as used herein refers to a limited hydration treatment prior to seed sowing of seeds to improve germination and seedling establishment, Figure 2 shows biochemical, physiological and biological processes occurring in seeds prior to germination. In the priming process, water availability is sufficient to initiate and influence early germination events, but not enough to allow rooting of young roots As such, the water availability for the seed is controlled and manipulated. Depending on the plant species and grower requirements, after the above controlled hydration, partial drying or complete drying can be further stored for short or long periods before seed sowing. Done.

  As used herein, the term “priming medium” refers to a solution (inorganic, eg, salt / nutrient, or organic, eg, PEG) or solid particulate system (eg, PEG) that provides controlled hydration with water. , Systems containing kaolin). Examples of such media are defined in Taylor GP et al. (1998, Seed Science Technology, 8: 245-256).

  The term “conditions that allow priming” refers to a medium composition (eg, osmotic) for inducing seed priming using any method known in the art for controlled moisture uptake. PEG solutions or other component solutions and solid water-absorbing agents, etc.); duration; temperature; light-dark mode; and aeration (eg, stirring, stirring, bubbling, etc.) suitable conditions.

  The term “treated plant” means that the heterologous DNA has been integrated into the plant genome, or has been integrated into its organelle, or is freely retained in the cytoplasm, or is episomal into the nucleus without being integrated. Regardless of whether it exists as a plant, it refers to a plant into which heterologous DNA has been introduced.

  The term “plant” is used herein in its broadest sense. The term includes, but is not limited to, any species of woody plant, herbaceous plant, perennial plant or annual. The term also refers to a large number of plant cells that are largely differentiated into structures that exist at any stage of plant development. Such structures include, but are not limited to, roots, stems, shoots, leaves, flowers, petals, fruits, any storage organ (eg, tubers, bulbs, corms, pseudostems, leaves, etc.). . The term “plant tissue” refers to the differentiated plant, including roots, shoots, leaves, pollen, seeds and tumors, and tissues present in cells in culture (eg, single cells, protoplasts, embryos, callus, etc.). Tissue and undifferentiated tissue are included. The plant tissue can be present in the plant or in organ culture, tissue culture or cell culture. The term “part of a plant” as used herein refers to a plant organ or plant tissue.

  The term “gene” refers to a nucleic acid (eg, DNA or RNA) sequence that comprises coding sequences necessary to produce an RNA or polypeptide. The term includes natural genes as well as human-crafted (synthetic) genes. A polypeptide can be encoded by a full-length coding sequence or by any portion thereof. The term “part thereof”, when used with respect to a gene, refers to a fragment of that gene, ranging from a few nucleotides in size to one nucleotide less than the complete gene sequence. Thus, a “nucleic acid sequence comprising at least a portion of a gene” can include a fragment of the gene or the entire gene.

  The term “gene” also encompasses the coding region of a structural gene and includes sequences found on both sides adjacent to the coding region at both the 5 ′ and 3 ′ ends over a distance of about 1 kb. A sequence located 5 'to the coding region and present in the mRNA is called a 5' untranslated sequence (5'UTR). The sequence located 3 ', ie downstream, of the coding sequence and present in the mRNA is referred to as the 3' untranslated sequence (3'UTR).

  The term “nucleic acid” as used herein refers to linear or branched, single-stranded or double-stranded RNA or DNA, or hybrids thereof. The term also includes RNA / DNA hybrids.

  The term “heterologous DNA” or the term “exogenous DNA” refers to a polynucleotide that is not present in its natural environment (ie, has been altered by the hand of man). For example, heterologous DNA includes a polynucleotide derived from one species and introduced into another species. Heterologous DNA is also for organisms that have been altered in some way (eg, mutated, added in multiple copies, linked to non-native promoter or enhancer sequences, etc.) Native polynucleotides are included. Heterologous DNA can include gene sequences of plant, bacterial and animal origin. Such gene sequences can include the cDNA form of the gene; the cDNA sequence can be in sense orientation (to produce mRNA) or an antisense RNA transcript that is complementary to the mRNA transcript. Can be expressed either in the antisense orientation. Heterologous plant genes are distinguished from endogenous plant genes in the following ways: Heterologous gene sequences are typically genes for proteins encoded by heterologous genes, or plant gene sequences in the chromosome and in nature. Regulatory elements that are not found to be associated (eg, promoters, etc.), or portions of chromosomes that are not found in nature (eg, genes that are expressed at a locus where the gene is not normally expressed) Linked to a nucleotide sequence that contains associated regulatory elements (eg, promoters, etc.).

  The term “construct” is used herein in its broadest sense to thereby refer to an artificially assembled or isolated nucleic acid molecule containing a heterologous DNA of interest. In general, a construct can include heterologous DNA (typically a gene of interest), optionally a marker gene, which can also be a gene of interest, and appropriate regulatory sequences. It should be understood that inclusion of regulatory sequences in the construct is optional as required. For example, such sequences may not be required in situations where host cell regulatory sequences will be used. The term construct includes a vector, but should not be understood as being limited thereto.

  The term “operably linked” indicates that a plurality of nucleic acid sequences are linked in a single nucleic acid fragment so that the function of one nucleic acid sequence is regulated by the other. For example, a promoter is operably linked to a coding sequence when the promoter is capable of regulating the expression of the coding sequence (ie, when the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

  The term “promoter element”, the term “promoter” or the term “promoter sequence” as used herein is located at the 5 ′ end of the protein coding region of a DNA polymer (ie, the protein coding region of a DNA polymer). DNA sequence in front of). Most promoters known in nature are located in front of the transcription region. The promoter functions as a switch that activates the expression of the gene. A gene is said to be transcribed or involved in transcription if it is turned on. Transcription involves the synthesis of mRNA from a gene. Thus, the promoter acts as a transcriptional regulatory element and also provides a site for initiating transcription of the gene into mRNA. A promoter may be derived from a natural gene in its entirety, or may be composed of different elements derived from various promoters found in nature, or even contain synthetic DNA segments. It will be appreciated by those skilled in the art that various promoters can direct gene expression in various tissues or cell types, at various stages of development, or in response to various environmental conditions. It is further appreciated that in most cases the exact boundaries of the regulatory sequences have not been fully defined, so that DNA fragments with some variation can have the same promoter activity. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. Various types of new promoters useful in plant cells are continually discovered; numerous examples can be found in Okamura JK and Goldberg RB (1989), Biochemistry of Plants, 15: 1-82.

  As used herein, the term “enhancer” refers to a DNA sequence that can stimulate promoter activity and is inserted to enhance the promoter's unique elements, or the level or tissue specificity of the promoter. Can be a heterogeneous element.

  The term “expression” as used herein refers to the production of a functional end product (eg, mRNA or protein).

Preferred Embodiments for Carrying out the Invention The present invention provides a method for introducing heterologous DNA into plant cells or tissues. The method of the present invention makes it possible to produce seeds and plants containing exogenous DNA by simple, easy to use, inexpensive, widely available and efficient means. According to the method of the present invention, the desired DNA is introduced into the seed, after which the plant is grown from the seed, which means that this is an efficient and costly and sometimes difficult plant cell for DNA introduction. Significantly increase the relief to the process.

  According to one aspect, the present invention is a method for introducing heterologous DNA into at least one cell of a plant seed embryo, the plant seed comprising heterologous DNA under conditions that allow priming. There is provided a method comprising contacting a priming medium containing a viral DNA construct, thereby obtaining a seed embryo containing heterologous DNA.

  According to some embodiments, the viral DNA construct comprises a viral gene or portion thereof that allows replication of the construct and asymptomatic expansion into adjacent plant cells.

  According to some embodiments, the method of the present invention further provides suitable conditions for allowing subsequent seed germination and growth to obtain a plant or part thereof comprising heterologous DNA. Including.

  The term “comprising heterologous DNA” when used with respect to a plant or seed refers to a plant or seed that contains at least one heterologous DNA in one or more of its cells. The term generally refers to a plant, plant structure, plant tissue, plant seed or plant cell that contains at least one heterologous DNA in at least one of its cells. The term includes primary cells into which DNA has been introduced, as well as cultures and plants derived from the cells regardless of the number of transfers. Not all offspring may be exactly identical in the content of the DNA due to intentional or accidental events. Mutant progeny that have the same functionality as screened in the original cell into which the DNA has been introduced are included in the definition of transformants.

  Introduction of heterologous DNA into the cell may be stable or transient. The term “transient” indicates that one or more exogenous polynucleotides are introduced into the cell in the absence of integration of the exogenous polynucleotide into the genome of the host cell. This type of DNA transfer is also sometimes referred to as “transient transformation”. Thus, the term “transient transformant” refers to a cell that has transiently taken up one or more exogenous polynucleotides. Transiently transformed cells are typically referred to as “non-transgenic” or “gene non-recombinant (non-GMO)”. In contrast, stable DNA transfer is referred to as “stable transformation” resulting in “stablely transformed” cells or tissues, and one or more exogenous polynucleotides are introduced and the cells Shows integration into the genome. The term “stable transformant” refers to a cell that has stably integrated one or more exogenous polynucleotides into genomic or organelle DNA (chloroplasts and / or mitochondria). A plant or part thereof comprising cells stably transformed with exogenous DNA is typically a “transgenic plant”, “transgenic plant cell”, or “transgenic seed” in the context of the present invention. Called.

  The viral DNA construct of the present invention is capable of systemic asymptomatic expansion in plants into which the viral DNA construct of the present invention has been introduced. As used herein, the term “systemic asymptomatic expansion” means that the viral vector does not induce the characteristic pathogenic symptoms of the virus, eg, leaves developing from embryonic cells Shows that it can spread to cells. The DNA construct can further be transmitted to plant progeny containing heterologous DNA. While not wishing to be bound by any particular theory or mechanism of action, such transfer may result from the expansion of viral DNA constructs into plant germ cells.

  According to some currently preferred embodiments, the DNA construct is a gemini as disclosed in International (PCT) Patent Application Publication No. WO2007 / 141790, which is incorporated herein by reference in its entirety. Based on the viral genetic components. The construct includes a heterologous polynucleotide sequence flanked by discrete nucleic acid sequences encoding a geminivirus replicase.

  According to other embodiments, the construct further comprises a polynucleotide sequence encoding a modified geminivirus coat protein (CP). According to an exemplary embodiment, the modified geminivirus coat protein has the amino acid sequence set forth in SEQ ID NO: 3. According to a further embodiment, the polynucleotide encoding a modified geminivirus coat protein comprises a mutation or deletion in the nucleotide encoding the N-terminal 100 amino acids.

  According to a further exemplary embodiment, the expression construct further comprises a polynucleotide sequence encoding a modified geminivirus V2 protein. According to an exemplary embodiment, the modified geminivirus V2 protein has the amino acid sequence set forth in SEQ ID NO: 4.

  According to a further exemplary embodiment, the expression construct further comprises a polynucleotide sequence encoding a modified geminivirus C4 protein. According to an exemplary embodiment, the modified geminivirus C4 protein has the amino acid sequence set forth in SEQ ID NO: 5. According to these embodiments, the modified geminivirus C4 protein comprises a mutation or deletion.

  It should be clearly understood that other viral-type constructs that have long heterologous polynucleotides and are capable of replication and asymptomatic expansion are also encompassed within the teachings of the present invention. As used herein, the term “long heterologous polynucleotide” refers to a polynucleotide of at least 1 kb in length (typically at least 5 kb in length, more typically 6 kb in length). .

  According to some exemplary embodiments, the DNA construct is based on tomato yellow leaf curl virus (TYLCV). Examples of such constructs are IL-60-BS as described in FIG. 2 (which has the nucleic acid sequence shown in SEQ ID NO: 2) and pIR-GUS (which is shown in SEQ ID NO: 8). Having a nucleic acid sequence).

  Geminivirus-type constructs used in accordance with the teachings of the present invention are introduced into seed germ cells, but not integrated into the cell's genome. Therefore, plants that grow from seeds containing heterologous DNA and plants that grow from the plants are called non-recombinant plants.

  The agricultural use of genetically modified plants is a matter of national debate and is not permitted by law or regulation in many countries. The main concerns that oppose the use of transgenic plants are inappropriate selection of transgenic lines (due to hidden harmful position effects), possible crossing with weeds and other crops, recombination There are concerns about further genomic alterations due to (especially when multiple copies of endogenous genes are added) and possible transmission of foreign genes to plants and soil microorganisms. The introduction of antibiotic resistance genes into food and the environment is also of great concern.

  Biosafety and environmental aspects can only be concluded when following actual, carefully controlled long-term field trials. Authorization to perform such experiments relies on evaluation based on rigorous laboratory data. One advantage of the method of the present invention stems from the discovery that TYCLV-based constructs are environmentally friendly and can be readily used for field testing of biosafety assessments. Geminiviruses are not seed contagious (Kashina et al., 2003, Phytoparasitica, 31: 188-199). The vector forms of IL-60-BS and pIR are not insect infectious even when a large number of insect vectors form a community in the plant. Therefore, TYCLV type constructs are very suitable for plant transformation.

  The presence of the heterologous DNA in the treated seed cells may be any suitable as known to those skilled in the art, regardless of whether the heterologous DNA is transiently expressed or stably integrated. This can be confirmed by using a simple method. Stable transformation of cells using Southern blot hybridization with nucleic acid sequences that can isolate genomic DNA and bind to one or more of the exogenous polynucleotides, or amplify the exogenous polynucleotide sequences It can be detected by using the polymerase chain reaction with appropriate primers. Expression of transforming DNA, eg, by an enzyme linked immunosorbent assay (ELISA) that detects the presence of a polypeptide encoded by one or more of the exogenous polynucleotides, or a protein encoded by the exogenous polynucleotide Can be detected by detecting the activity of.

  In the alternative, the exogenous DNA can include a marker. The marker provides for the identification and / or selection of cells, plants and / or seeds that express the marker. A marker can encode a product that, when expressed at a sufficient level, confers resistance to a selection agent. Such markers and their corresponding selection agents include herbicide resistance genes and herbicides, antibiotic resistance genes and antibiotics, and other chemical resistance genes and their corresponding chemicals However, it is not limited to these. Bacterial drug resistance genes include neomycin phosphotransferase II (nptII) which confers resistance to kanamycin, paromycin, neomycin and G418, and hygromycin phosphotransferase (hph) which confers resistance to hygromycin B. However, it is not limited to these. Resistance also includes amino acid synthesis including, but not limited to, imidazolinone compounds, sulfonylurea compounds, triazolopyrimidine compounds, glyphosate, cetoxydim, phenoxaprop, glufosinate, phosphinotricin, triazine compounds and bromoxynyl. It can be applied to herbicides from several groups, including inhibitors, photosynthesis inhibitors, lipid inhibitors, growth regulators, cell membrane disruptors, pigment-based inhibitors, seedling seedling growth inhibitors.

  A further type of marker is a marker whose expression can be detected by following a biochemical reaction that preferably develops color when given an appropriate substrate. Examples include GUS (β-glucuronidase) reporter system, luciferin-luciferase and green fluorescent protein (GFP) system.

  According to some embodiments, the DNA construct further comprises regulatory elements including, but not limited to, promoters, enhancers and termination signals.

  Very commonly used promoters include the nopaline synthase (NOS) promoter (Ebert et al., 1987, Proc. Natl. Acad. Sci. USA, 84: 5745-5749), octopine synthase ( OCS) promoter, caulimovirus promoter (eg, cauliflower mosaic virus (CaMV) 19S promoter (Lawton et al., 1987, Plant Mol Biol, 9: 315-324), CaMV 35S promoter (Odell et al., 1985, Nature, 313) : 810-812), and the 35S promoter of sesame mosaic virus, the light-inducible promoter derived from the small subunit of rubisco, the Adh promoter (Walker et al., 198 7, Proc. Natl. Acad. Sci. USA, 84: 6624-66280), sucrose synthase promoter (Yang et al., 1990, Proc. Natl. Acad. Sci. USA, 87: 4144-4148), R gene complex promoter (Chandler et al., 1989, Plant Cell, 1: 1175-1183), chlorophyll a / b binding protein gene promoter, etc. Other commonly used promoters are potato tuber ADPGPP Promoters for genes, sucrose synthase promoter, granule-bound starch synthase promoter, glutelin gene promoter, maize waxy promoter, Brittle gene promoter and Shr The nken2 promoter, the acid chitinase gene promoter, and the soybean gene promoter (15 kD, 16 kD, 19 kD, 22 kD, and 27 kD; Perdersen et al., 1982, Cell, 29: 1015-1026). / 18963.

  “3 ′ non-coding sequence” refers to a DNA sequence located downstream of the coding sequence, including polyadenylation recognition sequences and other sequences that encode regulatory signals that may affect mRNA processing or gene expression. included. The polyadenylation signal is usually characterized by affecting the addition of a polyadenylic acid region to the 3 'end of the mRNA precursor. The use of various 3 'non-coding sequences is exemplified by Ingelbrecht IL et al. (1989, Plant Cell, 1: 671-680).

  The methods of the present invention provide a desired property, such as (a) resistance to biological and abiotic stress conditions, such as resistance to insects, nematodes, viruses, bacteria, fungi and other pathogen organisms. Resistance to the disease caused, resistance to certain herbicides, (b) adaptation to poor conditions, eg salt tolerance, drought tolerance, cold tolerance and high temperature tolerance; (c) improved harvest characteristic traits (This includes pigmentation, firmness, type and content of ingredients (including sugars, volatiles, oils, fatty acids and / or acids), size, growth rate, fertile growth habits, rooting time, fruit appearance Including, but not limited to, maturation time); nutritional value or commercial value; (d) improved production of edible yield, or protein form, secondary metabolite form Improved production of secondary metabolites for both the biopharmaceutical and cosmetic industries, and (e) desired growth habits (including finite, semi-finite and infinite (e.g., Any polys whose presence and / or expression are noted, including for plants to obtain (for tomatoes), fertility and normal (eg, for corn), and obtaining plant vitality), etc. Can be used to introduce nucleotides. Introduced DNA can also provide gene silencing when the heterologous DNA is in the form of antisense, siRNA, dsRNA; or, but not limited to, fiber, wood, oil, resin, etc. Including industrial raw materials, as well as for generating other plant traits governed by genetic factors and / or interactions by genes and the environment.

  The expressed polynucleotide sequence can encode a molecule that will protect the plant from abiotic stressors (eg, sunshine, heat or cold). Examples include Myoxocephalus Scorpius (WO 00/00512) or M.C. Anti-freeze polypeptide derived from M. octodemspinosus, Arabidopsis thaliana transcriptional activator CBF1, glutamate dehydrogenase (WO97 / 12983, WO98 / 11240), calcium-dependent protein kinase gene) WO 98/26045), calcineurins (WO 99/05902), casein kinase derived from yeast (WO 02/052012), farnesyltransferase (WO 99/06580), ferritin (Deak M et al., 1999, Nature Biotechnology, 17: 192-196), Oxalate oxidase (WO99 / 04013), DREB1A factor (Kasuga M. et al., 19 9, Nature Biotech, 17: 276-286), inhibiting genes such as mannitol synthesis or trehalose synthesis (eg, trehalose phosphate synthase or trehalose phosphate phosphatase (WO 97/42326)) or trehalase ( WO 97/50561).

  The expressed polynucleotide sequence may be a metabolic enzyme used in the food and feed fields. Examples include phytase (GenBank accession number A19451) and cellulase.

  Expressed polynucleotide sequences directly attack pathogens, thereby activating host defenses or causing the accumulation of some metabolites or proteins, thereby causing viruses, fungi, insects, nematodes and others Can be resistant to pathogens and diseases. Examples include glucosinolate (protection from herbivores), chitinases or glucanases and other enzymes that destroy the cell walls of parasites, plants by microorganisms, or chemically, for example by salicylic acid, jasmonic acid or ethylene Ribosome inactivating protein (RIP) and other proteins of plant resistance and stress responses as induced when injured or attacked, or lysozyme from non-plant sources (eg T4 Lysozyme, or lysozyme from various mammals), insecticidal proteins (eg, Bacillus thuringiensis endotoxin), α-amylase inhibitors or protease inhibitors (cowage trypsin inhibitors) ), Lectins (eg, wheat germ agglutinin), siRNA, antisense RNA, RNAse or ribozymes. Further examples include Trichoderma harzianum chit42 endochitinase (GenBank accession number S78423) or N-hydroxylated multifunctional cytochrome P-450 (CYP79) accession (GenUb24 accession number) from Sorghum bicolor. Or nucleic acids encoding their functional equivalents.

  Resistance to pests, for example resistance to rice pests such as Nilaparvata lugens in rice plants, can be achieved by expressing snowdrop (Galanthus nivariis) lectin agglutinin (Rao et al., 1998, Plant J, 15 (4): 469-77).

  Expression of the synthesized cryIA (b) and cryIA (c) genes, which encode Lepidoptera-specific Bacillus thuringiensis δ-endotoxin, confers resistance to insect pests in various plants (Goyal RK et al., 2000, Crop Protection, 19 (5): 307-312).

  Additional genes that are suitable for pathogen defense include “polygalacturonase inhibitor protein” (PGIP), thaumatin, invertase and antimicrobial peptides such as lactoferrin (Lee T J et al., 2002, J Amer Soc Horticult Sci, 127 (2): 158-164). The expressed polynucleotide sequence can result in accumulation of chemicals, such as accumulation of tocopherols, tocotrienols or carotenoids. An example of such a polynucleotide is phytoene desaturase. Preferred is a nucleic acid encoding Narcissus pseudonarcissus phytoene desaturase (GenBank accession number X78815) or a functional equivalent thereof. The expressed polynucleotide sequence is used for the production of dietary supplements, for example for the production of polyunsaturated fatty acids (arachidonic acid, eicosapentaenoic acid or docosahexaenoic acid) by fatty acid elongases and / or fatty acid desaturases, or improved nutritional value. For example, a protein having a high content of essential amino acids (for example, a high methionine 2S albumin gene of Brazil nut) and the like. Preferred are polynucleotide sequences encoding: Bertholletia ecelsa high methionine 2S albumin (GenBank accession number AB044391), Physicomitrella patens δ-6-6 Acyl lipid desaturase (GenBank Accession No. AJ222980), Mortierella alpina δ-6-desaturase (Sakuradani et al., 1999, Gene, 238: 445-453), Caenorhabditis elegance (Caenorhabditis) δ-5-desaturase (Michaelson et al., 1998, FE S Letters, 439: 215-218), Caenorhabditis elegans A5-fatty acid desaturase (des-5) (GenBank Accession No. AF078796), Mortierella alpina δ-5-desaturase (Michaelson et al., JBC, 273). : 19055-19059), Caenorhabditis elegans δ-6-elongase (Beaudin et al., 2000, PNAS, 97: 6421-6426), Fiscomitrella patens δ-6-elongase (Zank et al., 2000, Biochemical Society Transactions, 28: 654-657) or functional equivalents thereof.

  Expression polynucleotide sequences are described, for example, by Hood EE et al. (1999, Curr Opin Biotechnol, 10 (4): 382-60) and Ma J K et al. (1999, Curr Top Microbiol Immunol, 236, 275-92). High quality proteins and enzymes for industrial purposes (eg enzymes, eg lipases) or pharmaceutical quality proteins and enzymes (eg antibodies, blood clotting factors, interferons, lymphokines, Colony stimulating factors, plasminogen activators, hormones or vaccines etc.) can be used. For example, recombinant avidin from chicken egg white and bacterial P-glucuronidase (GUS) can be produced on a large scale in transgenic corn plants (Hood et al., 1999, Adv Exp Med Biol, 127-47). ; Review).

  Expression polynucleotide sequences can also be used to obtain increased storage in cells that normally contain less storage protein or storage lipid, with the goal of increasing the yield of these substances (eg, Acetyl-CoA carboxylase). A preferred polynucleotide sequence is a polynucleotide sequence that encodes alfalfa (Medicago sativa) acetyl-CoA carboxylase (accase) (GenBank accession number L25042) or a functional equivalent thereof.

  Further examples of expressible polynucleotides include hepatitis B surface antigen (Kumar GBS et al., 2005, PLANTA, 222 (3): 484-493), herbicide resistance (Duke SO et al., Pest Management Science, 61 (21): 1-218), interferon (Edelbaum O et al., 1992, J. Interferon Res., 12: 449-453), T7-RNA polymerase (Zeitoune et al., 1997, Plant Science, 141: 59-65). included.

  Additional examples of polynucleotide sequences that can be expressed in the transformed plants of the present invention are described, for example, in Dunwell JM, 2000, J Exp Bot, 51: 487-96.

  Heterologous DNA transformed into plant cells in accordance with the teachings of the present invention can also be used to reduce (suppress) transcription and / or translation of target genes. Thus, a DNA construct can include heterologous DNA that results from a PTGS (post-transcriptional gene silencing) effect or a TGS (transcription silencing) effect, and thus a decrease in expression of an endogenous gene. Such a reduction can be achieved, for example, by the expression of antisense RNA or double stranded RNA, each having homology to the endogenous target gene to be repressed. Similarly, by expressing a suitable sense RNA, a decrease in the expression of an endogenous gene can be caused by means known as co-suppression (European Patent Application Publication No. 0465572). Particularly preferred is the expression of a small duplex interfering RNA (siRNA) to reduce gene expression of the target gene by RNA interference (RNAi). A method for inhibiting individual target genes using RNA having a duplex structure, in which case the target gene and the region of the RNA duplex have at least partial identity, It is known to those skilled in the art.

  Below are provided examples for applications in which reduced gene expression can be obtained by transforming plant seeds with appropriate heterologous DNA in accordance with the teachings of the present invention.

  Delayed fruit ripening or modified ripening phenotypes (long-term ripening, slower aging) can be achieved, for example, polygalacturonase, pectinesterase, β-1,4-glucanase (cellulase), β- A gene selected from the group consisting of galactanase (β-galactosidase), or a gene for ethylene biosynthesis (eg 1-aminocyclopropane-1-carboxylate synthase, adenosylmethionine hydrolase (SAMase), aminocyclopropane- 1-carboxylate deaminase, aminocyclopropane-1-carboxylate oxidase, etc.), genes for carotenoid biosynthesis, such as genes for prephytoene biosynthesis or phytoene biosynthesis (eg phytoene desaturase), and O-methyltransferase , Reed Decrease gene expression of carrier protein (ACP), elongation factor, auxin-inducible gene, cysteine (thiol) proteinase, starch phosphorylase, pyruvate decarboxylase, chalcone reductase, protein kinase, auxin-related gene, sucrose transporter, meristem pattern gene Can be achieved. Further advantageous genes are described, for example, in International (PCT) Patent Application Publication Nos. WO 91/16440, WO 91/05865, WO 91/16426, WO 92/17596, WO 93/07275 or WO 92/04456. Particular preference is given to reducing the expression of polygalacturonase in order to prevent plant and fruit (eg tomato) cell degradation and cocoon-like conditions. Nucleic acid sequences such as the tomato polygalacturonase gene (GenBank Accession No. x14074) or its homologous nucleic acid sequence can preferably be used for this purpose.

  Reduction of gene expression of genes encoding storage proteins has a number of advantages: for example, reducing the potential of allergens, or the composition or amount of other metabolites (eg oil or starch content) Etc.).

  Resistance to phytopathogenic organisms (eg spiders, fungi, insects, nematodes, protozoa, viruses, bacteria, etc.) and disease, growth of certain pathogenic organisms, survival, several developmental stages (eg hatching) ) Or by reducing gene expression of genes that are essential for reproduction. Such a reduction can result in complete inhibition of the above steps, or else a delay in the above steps. They can take the form of, for example, plant genes that allow the invasion of pathogenic organisms, but can also be homologous pathogenic organism genes. The transformed and expressed heterologous nucleic acid sequence (eg, double stranded RNA) is directed against the pathogenic organism so that the life cycle of the pathogenic organism is disrupted.

  Viral resistance can be achieved, for example, by reducing the expression of viral coat proteins, viral replicases and viral proteases. A large number of plant viruses and suitable target genes are known to those skilled in the art.

  Reduction of undesired allergenic or toxic plant components such as glucosinolate or patatin. Suitable target genes are described (among others in WO 97/16559). Preferred target genes for allergenic protein reduction are, for example, Tada Y et al. (1996), FEBS Lett, 391 (3): 341-345, or Nakamura R (1996), Biosci Biotechnol Biochem, 60 (8 ): 1215-1221.

  Delayed signs of aging. Suitable target genes are inter alia cinnamoyl-CoA: NADPH reductase or cinnamoyl alcohol dehydrogenase. Additional target genes are described (among others in WO95 / 07993).

  Increasing methionine content by decreasing threonine biosynthesis, for example, by reducing expression of threonine synthase (Zeh M et al., 2001, Plant Physiol, 127 (3): 92-802).

  Plant seeds are storage reserves of nutrients in zygote embryos resulting from sexual fertilization, or in structures called zygote embryos, cotyledons, endosperm or macrogametes due to asexual reproductive seed reproduction (no mating reproduction) As well as a complete reproducible unit containing all necessary items, generally consisting of storage reserves and protective seed coats surrounding the embryo. In nature, plant seed maturation is usually accompanied by the gradual loss of water over time to levels of water content between 5% and 35%. Once these low moisture levels are achieved, plant seeds can be stored for long periods of time.

  Germination of sexually zygotic and non-gametogenic plant seeds generally involves one or more environmental cues (eg, the presence of water, oxygen, optimal temperature or cold / hot treatment, and light) And its duration). Seeds begin with the uptake of water by dormant dry seeds (water-absorbing swelling), followed by various organisms that eventually lead to the elongation of the embryo along its axis as well as the development of offspring Germinate by a series of events that proceed through physical, biochemical and physiological events.

  The continuous process of seed germination can be divided into three stages. The first stage is called water absorption swelling and is characterized by a rapid initial uptake of water into the seed. Another important event that occurs in the first stage is the initiation, followed by the repair of damaged nuclear and mitochondrial DNA that may have occurred during the seed drying and / or maturation process. The protein synthesis promoted by mRNA is initiated.

  The second stage is characterized by a significant decrease in the water uptake rate (i.e. the water absorption swell has been completed). This is accompanied by activation or de novo synthesis of enzymes that are specialized to hydrolyze complex storage reserves of carbohydrates, proteins and lipids in embryos and cotyledons or macrogametes. Hydrolysis of these composite storage reserves provides the materials required for seed embryo respiration and growth.

  The third stage is characterized by a second rapid increase in water uptake rate. The water absorbed during the third phase is mainly used to initiate cell division of meristems at the roots and embryonic shoot apex, and for uptake into cells along the embryo axis. The Water taken up by embryonic axial cells exerts turgor pressure that causes axial cell elongation. The net effect is that the embryo extends through the seed coat to the root point. The shoot or root (young root) sticking out the seed coat means the completion of germination and the start of seedling growth and development.

  The rate and success for seed germination depends on various factors, for example, the residual effects of circular conditions in which the seed has developed and matured, the storage reserve compound synthesized during the seed maturation process It varies considerably depending on the quantity, the duration of storage, the quality of the storage environment (eg temperature and humidity), and the environmental conditions that dominate during the germination process. From a commercial point of view, it is desirable to reduce the risk of poor germination and to ensure that the seeds root and germinate quickly and uniformly.

  Commercial demands for optimal seed germination performance have led to the development of a process known in the art as “seed priming” for zygote seeds. This term is defined as a limited uptake of water (preferably followed by drying) that is sufficient to initiate an early event of germination but not enough to allow larval protrusion. Sometimes. Several principle techniques are used commercially to achieve seed priming. Regardless of the specific method used, the basic principle of seed priming is that (1) the germination preparatory stage is activated specifically and exclusively through controlling the water availability to the seed And (2) subsequently, the germination process initiated via the external priming process is stopped by a drying step or a partial drying step.

  Unexpectedly, the present invention now shows that inclusion of a viral DNA construct in the seed priming medium results in DNA uptake by the seed such that the DNA enters the seed cells. While not wishing to be bound by any particular theory or mechanism of action, this phenomenon can be attributed to viral elements that allow the introduction of heterologous DNA into seed cells.

  Any method for seed priming as known to those skilled in the art can be used in accordance with the teachings of the present invention. Priming may be performed using any of the techniques for controlled water uptake, ie, as described, for example, in Taylor A G et al. (1998, Seed Science Technology, 8: 245-256). Various using priming with inorganic, e.g. salt / nutrient or organic, e.g. PEG, or priming with solid particulate system or controlled hydration with water This can be done under temperature and aeration (eg, stirring, stirring, bubbling, etc.).

  The priming matrix is characterized by its effective osmotic potential. An effective osmotic potential typically reduces the water potential available for seed swell, which allows a limited amount of water to germinate without the juvenile roots actually protruding. Allowing or causing the seed to migrate to a level that is sufficient for the initial stage, i.e. to prime the seed. Seed germination occurs only when the water available to the seed reaches a potential sufficient for physiological development, which varies between plant species. Typically this value is comprised between 0 and 2 mPa. Many priming matrices that provide adequate osmotic potential continue to be used, including water, water with one or more solutes, and solid matrices. For example, the priming matrix can be an aerated solution of an organic osmotic material (eg, polyethylene glycol (PEG) (see US Pat. No. 5,119,598), glycerol, mannitol, etc.), or an inorganic salt (or An aerated solution of a combination of salts (eg, potassium phosphate and potassium nitrate, etc.). Alternatively, the seed can be primed using a solid matrix. The solid matrix material must have a large water holding capacity to allow the seeds to swell and absorb water. In this method, the priming matrix includes an absorbent medium (eg, clay, vermiculite, perlite, sawdust, corn cob and / or peat, etc.) for absorbing water and then transferring the water to the seed. (See, eg, US Pat. No. 4,912,874). The degree of hydration is controlled by changing the moisture content of the medium and the medium / seed ratio. Various methods are also known for water imbibing seeds in PEG6000 and vermiculite slurries, or in other matrices (eg, US Pat. No. 5,628,144). In yet another method, priming uses a semi-permeable membrane that mediates water transfer from solution to seed characterized by a given osmotic pressure (eg, US Pat. No. 5,873,197). In other methods, ultrasonic energy can be used to aid the priming process (eg, US Pat. No. 6,453,609). Optionally, various additives, chemicals and / or compounds can be included in the priming matrix, including surfactants, selective agents, fungicides, agents to modify osmotic potential, osmosis Pressure protectants, agents to aid drying or protect seeds during the drying period, agents to enhance seed processing, agents to extend shelf life, agents to increase coating and / or perfusion Included are drugs to increase seed germination. A fungicide can be included in the priming matrix (eg, thiram, captan, metalaxyl, pentachloronitrobenzene, phenaminosulfur, fungicide or other preservative). In addition, various growth regulators or hormones such as gibberellin or gibberellic acid, cytokinin, inhibitors of abscisic acid, 2- (3,4-dichlorophenoxy) triethylamine (DCPTA), potassium nitrate and ethephon are also used for priming It can be present in the matrix. Other drugs optionally used include glycerol, polyethylene glycol, mannitol, DMSO, Triton X-100, Tween-20, NP-40, ionic compounds, non-ionic compounds, surfactants and detergents, etc. included. Sufficient time to produce primed seed allows the pre-emergence metabolic process to take place in the seed to any level, including the level immediately before rooting To do. The time to produce the primed seed depends on the particular seed variety, its state or condition, and the moisture potential of the priming matrix. Typical moisture and medium moisture potentials for a given seed type are already generally known for some seeds, but a small sample of new seeds can be easily determined for osmotic potential and temperature. It is often best to determine what conditions of temperature, moisture potential and time result in proper water uptake of the seed and the resulting pre-emergence event. The temperature at which the priming process is performed can vary with the seed to be treated, but is typically between 18 ° C and 30 ° C. The primed seed can be retained in the priming matrix throughout germination as indicated by radicle rooting. Seeds produced by this method can be further dried (eg, as in US Pat. No. 4,905,411).

  Methods are known in the art to determine if seeds are primed. For example, optimization of priming can be performed by performing a germination assay (see, eg, Jeller H et al., 2003, Braz J Biol, 63: 61-68). In addition, molecular markers of germination and / or priming are known. For example, Job et al., 2000, Seed Biology: Advances and Applications (Black et al., CABI International, Wallingford, UK), pages 449-459; De Castro RD et al., 2000, Plant Physiol, 122: 3B; See, 2000, Seed Biology: Advances and Applications (Black et al., CABI International, Wallingford, UK), 221-251; and Gallardo K, et al., 2001, Plant Physiol, 126: 835-8.

  After priming, the seeds can be germinated or the primed seeds can be dried. Appropriate conditions for the drying process (temperature, relative humidity and time) vary depending on the seed and can be determined empirically (see, eg, Jeller et al. (2003, supra). . Drying the primed seed includes surface drying of the seed or, alternatively, drying the seed back to its original moisture content. The dried seed can be germinated immediately or stored under appropriate conditions. Germination conditions for various seeds are known. One factor in determining the appropriate germination conditions is the germination temperature threshold range, which is the temperature range for a species within which the seed of that species is at a given humidity level. And germinate with sufficient oxygen. Another factor is the germination humidity threshold range, which is the humidity range for a species within which the seed of that species germinates at a given temperature and with sufficient oxygen. As methods for empirically determining these conditions for any given seed and variety are known, germination temperature threshold ranges and / or germination humidity threshold ranges are known for various seeds. It has been.

  The method of the present invention significantly reduces the time required for introduction of heterologous gene (s) into plant cells, production of target plants, selection and propagation. The potential for somaclonal changes caused by tissue differentiation in culture is eliminated. When a geminivirus-type construct is used, the plant contains foreign DNA to contain any cell infected with benign microorganisms, yet the treated plant is not genetically modified. The method of the present invention facilitates the expression of the gene (s) of interest in any desired plant variety so long as the gene controlling the desired trait is known, so breeding Time and cost can be drastically reduced, thus required traits (eg resistance / resistance to biological / abiotic stress conditions, etc.) can be added, and yield and Properties can be improved for any given environment and market. Similarly, heterologous DNA introduced in accordance with the teachings of the present invention can cause silencing of a particular process (s) when so required (eg, development of greens in tomato fruits) Is desirable in Italy but not accepted in the United States).

  The following examples are presented in order to more fully illustrate some embodiments of the invention. However, the following examples should in no way be construed as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

Example 1: Priming using priming medium without foreign DNA Priming of tomato seeds in the presence of polyethylene glycol (PEG) was performed as previously described with some modifications (Khan, A A et al., 1992, Horticultural Reviews, Volume 14 (ed., J. Janick, New York: John Wiley), pages 131-181; Taylor G et al., 1998, Seed Science Technology, 8: 245-256).

50 tomato seeds per time were swollen with water in a solution composed of: 0.1 M Ca (NO ₃ ) ₂ , 0.1 M MES (4-morpholine ethanesulfonic acid), 15 mM MgCl ₂ and 25% (solution T2) or 20% (solution T3) of PEG 8000. The osmotic pressures of solution T2 and solution T3 were about 1.25 mPa and about 1.2 mPa, respectively. Untreated dry seeds (same seed lot) of the same cultivar were used as controls. The treatment was performed at a constant temperature of 20 ° C. with 12 hours of illumination per day and gently stirred for 4 or 6 days to obtain proper aeration. The seeds were then placed on 3M filter paper supplemented with 3 ml of sterile water and the germination rate was recorded daily. The data (FIG. 1) reveals a significant increase in germination uniformity of primed seeds compared to controls (successful priming).

Example 2: Introduction of IL-60 construct into seeds by using priming medium Tomato seeds were primed with a priming solution containing 25% PEG, IL-60-BS (SEQ ID NO: 2) and pIR -Germination as described in Example 1 above, in the presence of 20 μg, 40 μg and 60 μg of each of the DNA constructs of GUS (SEQ ID NO: 8). The construct is illustrated in FIGS. 2A and 2B, respectively. The construct is also described in International (PCT) Patent Application Publication Number WO2007 / 141790, which is incorporated herein by reference. Untreated dry seeds and seeds primed under the same conditions but without DNA construct served as controls.

Six days after germination, seedlings were transplanted into pots containing 10 liters of soil and placed in a 25 ° C. greenhouse. At 21 days after planting, DNA was extracted from the true leaf (third true leaf or above) and subjected to PCR using the following primers for the GUS assay:
Forward primer: ATTGATCAGCGTTGGTGGGA (SEQ ID NO: 6) and reverse primer: TGCGGTCGCGGATGGAAGATC (SEQ ID NO: 7) designed to amplify the entire gus gene present in the DNA construct of pIR-GUS. A positive PCR reaction confirmed that the GUS DNA sequence was present in the true leaf. These results indicate that the DNA construct of IL-60 type vector was introduced into seed cells using the priming procedure described above. The introduced DNA replicated, spread, and expressed throughout the plant system. An example of the results of such an assay is shown in FIG.

Example 3: Expression of foreign genes The IL-60 family DNA construct (IL-60-BS in combination with pIR-GUS) as detailed in Example 2 above is the pIR-GUS DNA construct. And facilitated the expression of foreign gus genes introduced by priming.

  Tomato leaves obtained from plants obtained as described in Example 2 above were stained for β-glucuronidase (GUS) according to Jefferson RA et al. (1987, EMBO J, 6: 3901-3907).

  Blue staining of leaf tissue clearly shows GUS protein expression (FIGS. 4A-4B).

Example 4: Expression of specific DNA from IL-60 vector under modified priming conditions Priming was performed at approximately 0.131 mPa (1.296 Atm., Christopher et al., Macromolecules, 2003, 36: 6888-6893. As in Example 2 using a basic priming solution containing 5% PEG and 75% dimethyl sulfoxide (DMSO) addition to obtain a final osmotic potential of It was. In this modified priming solution, IR-GUS DNA was introduced and replicated as shown by GUS expression as described in Examples 2 and 3 above.

  Since the foregoing description of these specific embodiments sufficiently clarifies the general nature of the present invention, those skilled in the art will apply undue experimentation to such specific embodiments by applying current knowledge. It can be easily modified and / or adapted for various applications without making and without departing from the generic concept, and thus such adaptation and modification are disclosed in the disclosed embodiments. Should be understood within the spirit and scope of equivalents of, and are intended to be understood within such scope. It should be understood that the expressions and terms used herein are for purposes of description and not limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.

SEQ ID NO: 1 is the sequence of IL-60.
SEQ ID NO: 2 is the sequence of IL-60-BS.
SEQ ID NO: 3 is the sequence of IL-60 coat protein.
SEQ ID NOs: 6 and 7 are synthetic primer sequences.
SEQ ID NO: 8 is the sequence of pIR-GUS.

Claims (29)

  A method for introducing heterologous DNA into at least one cell of a plant seed embryo, wherein the plant seed is contacted with a priming medium containing a viral DNA construct comprising the heterologous DNA under conditions allowing priming. Obtaining a seed embryo comprising heterologous DNA.   The method according to claim 1, further comprising a step of obtaining a plant body or a part thereof containing heterologous DNA by providing suitable conditions for subsequent seed germination and growth.   The method of claim 1, wherein the viral DNA construct comprises a viral gene or portion thereof that permits replication of the construct and asymptomatic expansion into neighboring plant cells.   4. The method of claim 3, wherein the DNA construct is a geminivirus type construct.   5. The method of claim 4, wherein the DNA construct comprises heterologous DNA flanked by discontinuous nucleic acid sequences encoding geminivirus replicase or replicase-associated protein.   6. The method of claim 5, wherein the DNA construct further comprises a polynucleotide sequence encoding a modified geminivirus coat protein (CP).   7. The method of claim 6, wherein the modified geminivirus coat protein comprises a mutation or deletion in the nucleotide encoding the N-terminal 100 amino acids.   6. The method of claim 5, wherein the DNA construct further comprises a polynucleotide sequence encoding a modified geminivirus V2 protein.   9. The method of claim 8, wherein the DNA construct further comprises a polynucleotide sequence encoding a modified geminivirus C4 protein.   6. The method of claim 5, wherein the DNA construct further comprises a bacterial polynucleotide sequence.   The method according to any one of claims 4 to 10, wherein the geminivirus is tomato yellow leaf curl virus (TYLCV).   The DNA construct is a geminivirus-type construct selected from the group consisting of IL-60 having the nucleic acid sequence shown in SEQ ID NO: 1 and IL-60-BS having the nucleic acid sequence shown in SEQ ID NO: 2. Item 12. The method according to Item 11.   The method of claim 1, wherein the DNA construct is an expression construct whereby heterologous DNA is expressed in plant cells.   14. The method of claim 13, wherein the DNA construct further comprises at least one regulatory element selected from the group consisting of an enhancer, a promoter, and a transcription termination sequence.   The method according to claim 1 or 2, wherein the DNA construct further comprises a marker for identifying seeds and plants containing the heterologous DNA.   2. The method of claim 1, wherein the heterologous DNA encodes a product selected from the group consisting of peptides, polypeptides, proteins and RNA molecules.   17. A method according to claim 16, wherein the peptide, polypeptide or protein is useful in the cosmetic or pharmaceutical industry.   17. The method of claim 16, wherein the RNA molecule is an inhibitory RNA selected from the group consisting of antisense RNA, dsRNA, and siRNA.   17. The encoded product provides a desirable agronomic trait selected from the group consisting of resistance to biological or abiotic stress, increased yield, increased harvest characteristics, and preferred growth patterns. the method of.   2. The method of claim 1, wherein the heterologous DNA is transiently expressed in at least one cell of the seed embryo and cells derived from the cell.   2. The method of claim 1, wherein the heterologous DNA is incorporated into the genome of at least one cell of the seed embryo and a cell derived from the cell.   The method of claim 1, wherein the priming medium has a water potential that allows moisture uptake of seeds but does not allow rooting of young roots.   24. The method of claim 22, wherein the priming medium is an aqueous solution.   24. The method of claim 22, wherein the priming medium is a soil matrix.   The seed produced by the method of claim 1, wherein the seed comprises a viral DNA construct comprising heterologous DNA.   26. A plant grown from seed according to claim 25 or a portion thereof, wherein the plant or a portion thereof comprises a viral DNA construct comprising heterologous DNA.   The method according to claim 1, wherein the seed is of monocotyledonous plant origin.   The method of claim 1, wherein the seed is of dicotyledonous origin.   The method of claim 1, wherein the seed is of cone-bearing origin.